Both inhibitory (vagal afferent) and excitatory (sympathetic or spinal afferent) reflexes are manifested during myocardial ischemia and reperfusion. Cardiac sympathetic afferent stimulation leads to symptoms of angina pectoris and potentially life threatening hemodynamic adjustments, including hypertension and arrhythmias that can exacerbate ischemia and infarction. During the last four cycles of this grant we have identified a number of chemical mediators released during ischemia that play an important role in activating ischemically sensitive cardiac spinal nerve endings to evoke reflex hypertension. These include bradykinin (BK), reactive oxygen species, protons, cyclooxygenase metabolites, serotonin (5HT), histamine, and most recently thromboxane A2 and endothelin. In the last funding cycle we observed that thromboxane and endothelin activate cardiac afferents during myocardial ischemia and reflexly elevates blood pressure. However, it is clear that other mediators signal these nerve endings since inhibition of the action or synthesis of a metabolite does not fully eliminate afferent activation during ischemia. In addition, we have identified roles for glutamate and nitric oxide in the rostral ventral lateral medulla (rVLM), which processes cardiac spinal afferent information, and, in turn, regulate sympathetic outflow. The current proposal further examines interactions between previously identified and new chemical mediators, including adenosine 5'- triphosphate (ATP) and opioids during myocardial ischemia, which act independently and interactively with other mediators to stimulate ventricular nerve endings. Thus, we have begun to investigate mechanisms of inhibitory chemical mediators in the heart that appear to modulate cardiac sympathetic afferent excitability during ischemia. With respect to central neural processing we are capitalizing on our previous observation that the external lateral parabrachial nucleus (elPBN) is activated by cardiac sympathetic afferent stimulation, to evaluate its role in processing input from the heart. This grant proposal thus proposes five sets of hypotheses designed to extend our past and preliminary studies of mechanisms of activation of cardiac afferents by 1) ATP, 2) opioid system, 3) interactions between excitatory and inhibitory peripheral chemical signals; and CNS processing of the cardiac afferents involving 4) excitation by glutamate and modulation by GABA through GABAB and GABAA receptors in elPBN neurons and 5) their projections to the rVLM. We will use combined whole animal reflex, cellular electrophysiological, microdialysis/HPLC measurement and pharmacological approaches in the proposed investigations. Additional knowledge derived from these studies will allow a better understanding of the mechanisms underlying the genesis of angina pectoris and reflex activation of the cardiovascular system during myocardial ischemia and reperfusion.